Reagents and methods for the beta-keto amide synthesis of a synthetic precursor to immunological adjuvant e6020

ABSTRACT

This invention relates to the synthesis for a precursor of E6020, compound 26, via a β-keto amide alcohol intermediate, compound 22. The synthesis reacts compound 22 with compound 25 and the resultant intermediate is oxidized to produce compound 26, the precursor to E6020. Compounds 22 and 25, and their crystalline forms, represent separate embodiments of the invention. The invention also relates to compounds of formulas (3) and (4) and processes for their preparation. 
     
       
         
         
             
             
         
       
     
     The β-keto amide alcohol intermediate compound 22 is a compound of formula (3). Compound 25 is a compound of formula (4).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/338,517, filed Dec. 18, 2008, which claims priority under 35U.S.C. §119 to U.S. provisional application Ser. No. 61/014,648 filedDec. 18, 2007, both of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the synthesis of precursors of theimmunological adjuvant E6020 via a β-keto amide intermediate. Theinvention also relates to intermediate compounds in that synthesis and,for two compounds, their crystalline forms.

BACKGROUND OF THE INVENTION

Vaccines have proven to be successful methods for the prevention ofinfectious diseases. Generally, they are cost effective, and do notinduce antibiotic resistance to the target pathogen or affect normalflora present in the host. In many cases, such as when inducinganti-viral immunity, vaccines can prevent a disease for which there areno viable curative or ameliorative treatments available.

Vaccines function by triggering the immune system to mount a response toan agent, or antigen, typically an infectious organism or a portionthereof that is introduced into the body in a non-infectious ornon-pathogenic form. Once the immune system has been “primed” orsensitized to the organism, later exposure of the immune system to thisorganism as an infectious pathogen results in a rapid and robust immuneresponse that destroys the pathogen before it can multiply and infectenough cells in the host organism to cause disease symptoms. The agent,or antigen, used to prime the immune system can be the entire organismin a less infectious state, known as an attenuated organism, or in somecases, components of the organism such as carbohydrates, proteins orpeptides representing various structural components of the organism.

In many cases, it is necessary to enhance the immune response to theantigens present in a vaccine in order to stimulate the immune system toa sufficient extent to make a vaccine effective, i.e., to conferimmunity. Many protein and most peptide and carbohydrate antigens,administered alone, do not elicit a sufficient antibody response toconfer immunity. Such antigens need to be presented to the immune systemin such a way that they will be recognized as foreign and will elicit animmune response. To this end, additives (adjuvants) have been devisedwhich stimulate, enhance and/or direct the immune response toward aselected antigen.

The best known adjuvant, Freund's complete adjuvant, consists of amixture of mycobacteria in an oil/water emulsion. Freund's adjuvantworks in two ways: first, by enhancing cell and humoral-mediatedimmunity, and second, by blocking rapid dispersal of the antigenchallenge (the “depot effect”). However, due to frequent toxicphysiological and immunological reactions to this material, Freund'sadjuvant cannot be used in humans.

Another molecule that has been shown to have immunostimulatory oradjuvant activity is endotoxin, also known as lipopolysaccharide (LPS).LPS stimulates the immune system by triggering an “innate” immuneresponse, a response that has evolved to enable an organism to recognizeendotoxin (and the invading bacteria of which it is a component) withoutthe need for the organism to have been previously exposed. While LPS istoo toxic to be a viable adjuvant, molecules that are structurallyrelated to endotoxin, such as monophosphoryl lipid A (“MPL”) have beentested as adjuvants in clinical trials. Both LPS and MPL have beendemonstrated to be agonists to the human toll-like receptor-4 (TLR-4).Currently, however, the only FDA-approved adjuvant for use in humans isthe aluminum persulfate salt, Alum, which is used to “depot” antigens byprecipitation of the antigens. Alum also stimulates the immune responseto antigens.

E6020 is a potent TLR-4 receptor agonist, and thus is useful as animmunological adjuvant when co-administered with antigens such asvaccines for bacterial and viral diseases. For example, E6020 may beused in combination with any suitable antigen or vaccine component,e.g., an antigenic agent selected from the group consisting of antigensfrom pathogenic and non-pathogenic organisms, viruses, and fungi. As afurther example, E6020 may be used in combination with proteins,peptides, antigens and vaccines which are pharmacologically active fordisease states and conditions such as smallpox, yellow fever, cancer,distemper, cholera, fowl pox, scarlet fever, diphtheria, tetanus,whooping cough, influenza, rabies, mumps, measles, foot and mouthdisease, and poliomyelitis, as well as viral diseases such as herpes andherpes-related diseases and hepatitis and hepatitis-related diseases.When used as a vaccine, E6020 and the antigen are each present in anamount effective to elicit an immune response when administered to ahost animal, embryo, or ovum being vaccinated therewith.

With their ability to stimulate a more robust antibody response thanwith an antigen alone, compounds such as E6020 are importantimmunological compounds. There is a need to develop synthetic methodsfor preparing compounds such as E6020, and their synthetic precursors,which can be co-administered with antigens in vaccines. New syntheticmethods involve new compounds as intermediates and new reactions asmethod steps. The invention provides an improved method for synthesizingintermediates and precursors for TLR-4 receptor agonists, such as E6020.

SUMMARY OF THE INVENTION

This invention relates to a new synthesis, intermediates and precursorsleading to E6020 precursor Compound 26 via a β-keto amide intermediateCompound 22. The synthesis of the invention starts from Compound 14 toprepare Compound 22, which is then reacted with Compound 25 in thepreparation of Compound 26, the penultimate precursor to theimmunological adjuvant E6020. Compounds 22 and Compound 25, and theircrystalline forms, represent separate embodiments of the invention.

In another embodiment, the invention relates to a compound of formula(3):

wherein R¹, R² and R³ are in each occurrence independently a C₅-C₁₅alkyl group, a C₅-C₁₅ alkenyl group, or a C₅-C₁₅ alkynyl group. Theinvention also relates to a process for preparing compounds of formula(3). The β-keto amide intermediate Compounds 22 is a compound of formula(3).

Another embodiment of the invention is a compound of formula (4):

wherein R⁴ is in each occurrence independently a protecting group, suchas a C₁-C₆ alkyl group, a C₃-C₅ alkenyl group, an aryl group, a benzylgroup or another suitable protecting group; and each of R⁵ and R⁶ is,independently in each occurrence, a C₁-C₆ alkyl group or, taken togetherwith the nitrogen to which they are attached, form a 5- or 6-memberedheterocyclic ring. The invention also relates to a process for preparingcompounds of formula (4). Compound 25 is a compound of formula (4).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overview of the β-keto amide synthesis of E6020 accordingto the invention.

FIG. 2 is the powder X-ray diffraction pattern of crystalline Compound22.

FIG. 3 is the DSC thermogram of crystalline Compound 22.

FIG. 4 is the powder X-ray diffraction pattern of crystalline Compound25.

FIG. 5 is the DSC thermogram of crystalline Compound 25.

FIG. 6 is an ORTEP drawing of crystalline Compound 25 with variousatomic labeling.

FIG. 7 is the crystal packing diagram of crystalline Compound 25 alongthe c-axis.

FIG. 8 is a simulated powder X-ray diffraction pattern for crystallineCompound 25.

DETAILED DESCRIPTION OF THE INVENTION

U.S. patent application Ser. No. 11/477,936 “Compounds For PreparingImmunological Adjuvant” filed Jun. 30, 2006 (published Apr. 12, 2007 asUS 2007-0082875 A1) describes the synthesis of E6020 and is incorporatedherein by reference. That synthesis proceeds through a phosphoric acidester ureido dimer, Compound 19. Schemes 1-3 of US 2007-0082875 A1depict the synthesis of E6020 via Compound 19. The starting materialsshown in Scheme 1 are ER-028694 (1,3-decanediol; commercially availablefrom vendor Mitsui & Co. (US), New York, N.Y.; manufacturer Nippon FineChemicals) and ER-807277 (4-Oxazolemethanol, 4,5-dihydro-2-phenyl-,(4R)-propanediol; commercially available from CatalyticaPharmaceuticals, Boonton, N.J.). The penultimate precursor to E6020 isCompound 26.

This invention relates to a new synthesis, intermediates and precursorsleading to E6020 precursor Compound 26 via a β-keto amide intermediateCompound 22. The synthesis of the invention starts from Compound 14 toprepare Compound 22, which is then reacted with Compound 25 in thepreparation of Compound 26, the precursor to the immunological adjuvantE6020. These steps of the synthesis of the invention, the preparation ofthe β-keto amide intermediate (3) followed by a condensation of theβ-keto amide (3) with a urea di-phosphoramidite (4), are shown in Scheme4 and described below in detail. Compounds 22 and 25, and theircrystalline forms, represent separate embodiments of the invention.

Obtaining a crystalline form of a compound, such as Compound 22 or 25,is extremely useful in drug development. Solid state forms (crystallineor amorphous) of a compound can have different physical and chemicalproperties, for example, solubility, stability, or the ability to bereproduced. These properties often permit the optimization ofmanufacturing processes, particularly where a crystalline intermediateis obtained. In multi-step syntheses, such as those described herein,intermediates are prepared and unwanted by-products or impurities can becarried forward from earlier steps. Often filtration, separation, and/orpurification steps are introduced to remove unwanted by-products orimpurities. Introducing such steps not only can increase manufacturingcosts but can also decrease the overall yield of the synthesis. Having acrystalline intermediate within a multi-step synthesis can address theseproblems. A crystalline intermediate provides certain advantages—a highpurity intermediate can reduce the need for other purification steps andreduce the cost of the synthetic process.

Preparation of the β-Keto Amide Alcohol Intermediate (3).

The synthesis of Compound 26, and similar compounds, first involvesreaction of an α-hydroxyl amine of formula (1) with a compound offormula (2) to form a β-keto amide of formula (3) under suitablereaction conditions. This is shown in Scheme 5.

Preparing compound (3) according to Scheme 5 may be achieved usingdifferent preparations. The groups R¹, R² and R³ are as defined below.Because R¹, R² and R³ may vary independently one from the other,compound (3) may have symmetrical or asymmetrical groups at R¹, R² andR³. In compound (2), X is a suitable leaving group such as, for example,OH, Cl, F, an imidazolidyl, a carbonate, and an ester. Preferred leavinggroups include OH, Cl, F, imidazolidyl, trimethyl acetoxy, ethylcarbonate, methyl carbonate, isobutyl carbonate or a group of theformula Z:

where R⁷ and R³ in formula (2) are identical such that the compound offormula (2) is a symmetrical beta ketoester anhydride. Such anhydridescan be obtained from the condensation of two identical beta-ketoacidmolecules.

In one preparation, compound (3) could be obtained from the condensationof compound (1) with compound (2) where X is imidazolide. The latter isobtained from the activation of compound (2) where X is OH (carboxylicacid) with CDI, (carbonyldiimidazole), reagent in a polar aproticsolvent such as acetonitrile.

In a second preparation, compound (3) could also be obtained from thecondensation of compound (1) with compound (2) where X is OH (carboxylicacid) in the presence of an amide bond coupling reagent like HBTU,(O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), or other coupling reagents in the same family anda tertiary amine base (e.g. Hünig's base, N,N-diisopropylethylamine) ina solvent such as DMF or CH₂Cl₂. In addition carbodiimide reagents suchas EDC (N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride),can be used to activate compound (2) where X is OH (carboxylic acid).

A third preparation to prepare compound (3) is the condensation ofcompound (1) with compound (2) where X is F (acyl fluoride) in thepresence of a tertiary amine base (e.g. Hünig's base) in a solvent likeCH₂Cl₂. Compound (2) where X is F (acyl fluoride) can be generated fromthe activation of compound (2) where X is OH (carboxylic acid) withTFFH, (fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate),reagent in a solvent such as CH₂Cl₂.

Compound (3) could also be obtained via a fourth preparation from thecondensation of compound (1) with compound (2) where X is Cl (acylchloride) in the presence of a tertiary amine base (e.g. Hünig's base)in a solvent such as CH₂Cl₂. Compound (2) when X is Cl (acyl chloride)can be generated from the activation of compound (2) where X is OH(carboxylic acid) with oxalyl chloride reagent in a solvent such asCH₂Cl₂.

A fifth preparation can prepare compound (3) from the condensation ofcompound (1) with alkanoic acid carbonic acid anhydride compound (2)where X is a carbonate in the presence of a tertiary amine base (e.g.Hünig's base) in a solvent such as CH₂Cl₂. The latter is obtained fromthe activation of (2) where X is OH (carboxylic acid) with ethylchloroformate reagent in the presence of a tertiary amine base (e.g.triethylamine) in a solvent such as CH₂Cl₂.

A sixth preparation can prepare compound (3) from the condensation ofcompound (1) with mixed anhydride compound (2) where X is an ester inthe presence of a tertiary amine base (e.g. Hünig's base) in a solventsuch as CH₂Cl₂. The latter is obtained from the activation of (2) whereX is OH (carboxylic acid) with pivaloyl chloride reagent in the presenceof a tertiary amine base (e.g. triethylamine) in a solvent such asCH₂Cl₂.

Compound (3) could also be obtained via a seventh preparation wherecompound (2) X is OH (carboxylic acid) is first converted into asymmetrical anhydride by the action of oxalyl chloride reagent and atertiary amine base (e.g. triethylamine) in a solvent such as CH₂Cl₂.The resultant anhydride is condensed with compound (1) in the presenceof a tertiary base (e.g. triethylamine) in a solvent such as CH₂Cl₂.

As shown in Example 1 below, a compound of formula (1) may be preparedby hydrogenating a compound of formula Y:

wherein R¹ and R² are as defined above. The hydrogenation may be carriedout in any suitable solvent, e.g. ethanol, isopropanol and others knownin the art, but preferably is done in isopropanol.

In formulas (1), (2), and (3), each of R¹, R² and R³ may be,independently, for each occurrence a C₅-C₁₅ alkyl group, a C₅-C₁₅alkenyl group, or a C₅-C₁₅ alkynyl group. The alkyl group, the alkenylgroup, and the alkynyl group may be substituted or unsubstituted,straight or branched and is preferably a straight chain. In a preferredembodiment, R¹ is a C₅-C₁₂ alkyl group, a C₅-C₁₂ alkenyl group, or aC₅-C₁₂ alkynyl group and more preferably a C₅-C₉ alkyl group, a C₅-C₉alkenyl group or a C₅-C₉ alkynyl group, while R² and R³ each is,independently for each occurrence, a C₇-C₁₄ alkyl group or a C₇-C₁₄alkenyl group and more preferably a C₉-C₁₃ alkyl group or a C₉-C₁₃alkenyl group. Preferably, R¹ is a C₇ alkyl group, most preferablyunsubstituted n-heptyl group, and R² and R³ are both a C₁₁ alkyl group,most preferably an unsubstituted n-undecyl group.

The alkyl, alkenyl, alkynyl groups mentioned for the various groups ofR¹, R², and R³ above discussed with regard to the invention may besubstituted or unsubstituted. Examples of substituents include, but arenot limited to, halo substituents, (e.g. F, Cl, Br, and I); a C₁-C₆alkoxy group, (e.g, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, and the like); a C₁-C₆haloalkyl group, (e.g., —CF₃, —CH₂CF₃, —CHCl₂, and the like); a C₁-C₆alkylthio; an amide; —NO₂; and —CN.

Unless otherwise specified, the alkyl, alkenyl, and akynyl groups may bestraight chains or branched with straight chains being generallypreferred.

The β-keto amide alcohol compounds of formula (3) are novel compoundsand are useful as, inter alfa, intermediates in the preparation ofcompounds such as E6020. Compounds of formula (3) are a separateembodiment of the invention. β-keto amide alcohol compounds having thestereochemistry shown in formula (3a) are particularly preferred.

In the synthesis of E6020, the β-keto amide alcohol is Compound 22.Compound 22 and its crystalline form are also separate embodiments ofthe invention. The synthesis of Compound 22 starting from Compound 14 isdescribed below in Example 1, and the solid state characterization ofcrystalline 22 is described below in Example 2.

Preparation of a Urea Di-Phosphoramidite (4).

The urea di-phosphoramidite compounds of formula (4) represent anothernovel intermediate compound in the β-keto amide alcohol synthesis ofE6020. Therefore compounds of formula (4) are a separate embodiment ofthe invention. The urea di-phosphoramidite compounds of formula (4) maybe prepared by reacting a 1,3-bis(2-hydroxyethyl)urea with twophosphorodiamidite compounds of formula (7) as shown in Scheme 6, whereR⁴, R⁵, and R⁶ are as described below.

In some embodiments, reaction of 1,3-bis(2-hydroxyethyl)urea with aphosphorodiamidite is effected by reacting the bis-hydroxy compound witha suitable activating agent such as is known to those skilled in theart, and then adding the phosphorodiamidite compound to the reactionmixture. In some embodiments, the activating agent is chosen from thegroup consisting of (1H)-tetrazole and pyridinium trifluoroacetate. Insome embodiments, the activating agent is pyridinium trifluoroacetate.In some embodiments, the reaction of Scheme 6 is run in a solventcomprising a solvent selected from the group consisting of acetonitrile,dichloromethane, dichloroethane, and tert-butylmethyl ether. In someembodiments, the reaction of Scheme 6 is run in acetonitrile as asolvent.

Condensation of 13-Keto Amide Alcohol (3) with Urea Di-Phosphoramidite(4).

As shown in Scheme 4 above, the synthesis of Compound 26 involves acondensation reaction of a β-keto amide alcohol of formula (3) with aurea di-phosphoramidite compound of formula (4) to form a β-keto amideurea phosphite compound of formula (5) which may then be oxidized toform a β-keto amide urea phosphate compound of formula (6). This isshown in Scheme 7 below.

In formulas (4), (5), and (6), R¹, R² and R³ are the same as describedabove, including their preferred embodiments, and are independent of oneanother. In each occurrence, R¹, R² and R³ may be the same or different.In other words, a compound of formula (6) may have the same or differentalkyl, alkenyl and/or alkynyl groups at each R¹, R² or R³ position. EachR⁴ group may independently be a protecting group, including but notlimited to the following groups: an alkyl or substituted alkyl groupsuch as methyl, ethyl, isopropyl, tert-butyl, and the like, preferably aC₁-C₆ alkyl group; an alkenyl or substituted alkenyl group such asallyl, 2-methyl propenyl, butenyl, and the like, preferably a C₃-C₅alkenyl group; an alkynyl, preferably a C₃-C₅ alkynyl group; acycloalkyl group such as cyclohexyl; a 2-substituted ethyl group such as2-cyanoethyl, 2-cyano-1,1-dimethylethyl, 2-(trimethylsilyl)ethyl, andthe like; a haloethyl group such as 2,2,2-trichloroethyl,2,2,2-trichloro-1,1-dimethylethyl, 2,2,2-tribromoethyl, and the like; abenzyl or substituted benzyl group such as benzyl, 4-nitrobenzyl,4-chlorobenzyl, and the like; an aryl or substituted aryl group such asphenyl, 4-nitrophenyl, 4-chlorophenyl, 2-chlorophenyl, 2-methylphenyl,2,6-dimethylphenyl, 2-bromophenyl, and the like; and a silyl group suchas trimethylsilyl and the like. Particularly preferred protecting groupsfor R⁴ are methyl, ethyl, tert-butyl, allyl, 2-methyl propenyl, butenyl,2-cyanoethyl (NCCH₂CH₂—), 2-(trimethylsilyl)ethyl ((CH₃)₃SiCH₂CH₂—), and2,2,2-trichloroethyl (Cl₃CCH₂—). Like R¹, R² and R³, each occurrence ofR⁴ may vary from one another within the definition of R⁴.

R⁵ and R⁶ are in each occurrence independently a C₁-C₆ alkyl group, aC₃-C₆ alkenyl group or a C₃-C₆ alkynyl group, wherein said alkyl,alkenyl, and alkynyl group may be substituted or unsubstituted, or,taken together with the nitrogen to which they are attached, form a 5-or 6-membered heterocyclic ring. The heterocyclic ring may includeadditional heteroatoms, e.g., N, O, and/or S; may be saturated orunsaturated, and may be unsubstituted or substituted. Examples ofsubstituents include, but are not limited to, halo substituents, (e.g.F, Cl, Br, and I); a C₁-C₆ alkoxy group, (e.g, —OCH₃, —OCH₂CH₃,—OCH(CH₃)₂, and the like); a C₁-C₆ haloalkyl group, (e.g., —CF₃,—CH₂CF₃, —CHCl₂, and the like); a C₁-C₆ alkylthio; a —NO₂ group; and a—CN group. Suitable heterocyclic groups include, but are not limited to,piperidyl, morpholinyl, thiomorpholinyl, pyrrolidyl, and the like. Thealkyl, alkenyl, and alkynyl groups of R⁴, R⁵ and R⁶ may be substitutedor unsubstituted, straight or branched. Examples of substituentsinclude, but are not limited to, halo substituents, (e.g. F, Cl, Br, andI); a C₁-C₆ alkoxy group, (e.g, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, and thelike); a C₁-C₆ haloalkyl group, (e.g., —CF₃, —CH₂CF₃, —CHCl₂, and thelike); a C₁-C₆ alkylthio; a —NO₂ group; and a —CN group. In a preferredembodiment, R⁴ is a C₃-C₅ alkenyl group and R⁵ and R⁶ are eachindependently a branched C₁-C₆ alkyl group. Preferably, R⁴ is an allylgroup and R⁵ and R⁶ are isopropyl groups.

In some embodiments the compound of formula (3) is reacted with thecompound of formula (4) by adding solvent to the mixture of compound offormula (3) and the compound of formula (4), stiffing until all solidsare dissolved, and adding acetic acid to the mixture. In someembodiments, the solvent is anhydrous and may be a co-solvent mixture.In some embodiments, the co-solvent mixture comprises acetonitrile and ahydrocarbon solvent and in a preferred embodiment, the solvent may be,for example, a mixture of anhydrous acetonitrile and anhydrous heptane.In some embodiments the co-solvent mixture comprises heptane andacetonitrile. In some embodiments, the weight of heptane in theco-solvent mixture is between 4 and 6 times the weight of the compoundof formula (3). In some embodiments, the weight of heptane in theco-solvent mixture is between 4.5 and 5.5 times the weight of thecompound of formula (3). In some embodiments the weight of acetonitrilein the co-solvent mixture is between 1 and 2 times the weight of thecompound of formula (3). In some embodiments the weight of acetonitrilein the co-solvent mixture is between 1.2 and 1.5 times the weight of thecompound of formula (3). In some embodiments, the weight of acetic acidused is between 1 and 2 molar equivalents of the amount of the compoundof formula (3) used. In some embodiments, the amount of acetic acid usedis between 1.2 and 5 molar equivalents of the amount of the compound offormula (3) used. In some embodiments, the temperature of the reactionmixture is maintained at about 20-25° C. during the addition of aceticacid.

In some embodiments, the compound of formula (5) formed by the method ofScheme 7 is oxidized to the compound of formula (6) by an oxidizingagent. In some embodiments, the oxidizing agent is hydrogen peroxide,Oxone® oxidant, mCPBA (meta-chloro perbenzoic acid), and the like. Insome embodiments the hydrogen peroxide is 30% (wt/wt) H₂O₂ in water. Insome embodiments, the reaction mixture containing the formed compound offormula (5) is diluted with additional heptane before the oxidationstep. In some embodiments, the weight of the additional heptane added isbetween 5 and 10 times the weight of the compound of formula (3) used.In some embodiments, the weight of the additional heptane added is about8 times the weight of the compound of formula (3) used. In someembodiments, the reaction mixture containing the formed compound offormula (5) is cooled to about −5-10° C. after adding the additionalheptane and before adding the oxidizing agent. In some embodiments, thereaction mixture containing the formed compound of formula (5) is cooledto about 0-5° C. after adding the additional heptane and before addingthe oxidizing agent. In some embodiments, the temperature of thereaction mixture is maintained at about −5-10° C. after adding theoxidizing agent. In some embodiments, the temperature of the reactionmixture is maintained at about 0-2° C. after adding the oxidizing agent.In some embodiments, the reaction is stirred at a temperature about−5-10° C. following addition of the oxidizing agent until the reactionis complete. In some embodiments, the reaction is stirred at atemperature about −1-2° C. following addition of the oxidizing agentuntil the reaction is complete. In some embodiments, the reaction ismonitored by HPLC to determine completion. In some embodiments, thereaction is quenched with sodium thiosulfate pentahydrate to destroyexcess peroxides after the reaction is complete.

In the synthesis of Compound 26, the compound of formula (4) is Compound25. Compound 25 and its crystalline form are also separate embodimentsof the invention. The synthesis of Compound 25 starting from thedihydroxy urea Compound 24 is described below in Example 4, and thesolid state characterization of crystalline Compound 25 is describedbelow in Example 5.

Example 6 below describes the preparation of E6020 precursor Compound 26(a species of the compounds of formula (6) where R¹ is n-heptyl, R² andR³ are both n-undecyl, and R⁴ is allyl) from the reaction of Compound 22and Compound 25.

The synthetic method described herein may be adapted to the preparationof any of all possible stereoisomers of Compound 26 and correspondinglyE6020, e.g., compounds (A) and (B) respectively below having the generalstructures:

While the examples below disclose the preparation of a particularstereoisomer, methods for preparing other stereoisomers of Compound 26are considered to fall within the scope of the present invention. Thesynthetic method of the invention is well suited to provide purestereoisomers for which the initial stereochemistry of the startingmaterial (3) is defined and used in a 2 equivalent ratio to the ureadi-phosphoramidite (4) as shown in Scheme 7 (above). This method wouldallow one to prepare four pure stereoisomers including E6020 with a(R,R,R,R) stereochemistry at positions 1, 6, 22, 27; ER-824156(S,S,S,S); ER-804053 (R,S,S,R); and ER-824095 (S,R,R,S). To prepare theother stereoisomers (ER-826685 (R,R,S,R); ER-824887 (R,R,R,S); ER-826682(R,R,S,S); ER-827905 (R,S,R,S); ER-826683 (R,S,S,S); ER-804097(S,S,R,S)) one would obtain a mixture of products for which one woulduse 1 equivalent of starting material (3) with a defined stereochemistryat positions 2 and 7 while a second equivalent of starting material (3)would have a differently defined stereochemistry at positions 2 and 7.The resultant reaction (using Scheme 7) would provide 3 differentstereoisomers of compound (6) that can be separated by columnchromatography and structurally determined by ¹H-NMR and HPLC comparisonto authentic stereoisomers previously prepared by the first syntheticmethod described above and a separate method as described in U.S. Pat.No. 6,290,973. U.S. Pat. Nos. 6,551,600; 6,290,973; and 6,521,776, whichdisclose other synthetic routes to E6020 and related compounds, providehelpful background information on preparing certain reagents andstarting materials and are incorporated herein by reference.

EXAMPLES

In the examples below, when the term “inerted” is used to describe areactor (e.g., a reaction vessel, flask, glass reactor, and the like) itis meant that the air in the reactor has been replaced with anessentially moisture-free or dry, inert gas (such as nitrogen, argon,and the like). The term “equivalent” (abbreviation: eq) as used hereindescribes the stoichiometry (molar ratio) of a reagent or a reactingcompound by comparison to a pre-established starting material. The term“weight” (abbreviation: wt) as used herein corresponds to the ratio ofthe mass of a substance or a group of substances by comparison to themass of a particular chemical component of a reaction or purificationspecifically referenced in the examples below. The ratio is calculatedas: g/g, or Kg/Kg. The term “volume” (abbreviation: vol) as used hereincorresponds to the ratio of the volume of a given substance or a groupof substances to the mass or volume of a pre-established chemicalcomponent of a reaction or purification. The units used in the equationinvolve matching orders of magnitude. For example, a ratio is calculatedas: mL/mL, mL/g, L/L or L/Kg. The following abbreviations are usedherein:

Abbreviation Chemical ACN acetonitrile Boc tert-butoxycarbonyl CDIcarbonyldiimidazole DMAP 4-(dimethylamino)pyridine DMF dimethylformamideEDC (N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride) EtOHethanol Et₃N triethylamine Fmoc 9-fluorenylmethoxycarbonyl NaHMDS sodiumhexamethyldisilazide HOAc acetic acid IPA isopropyl alcohol iPrisopropyl Pd/C palladium on carbon Pd(PPh₃)₄tetrakis(triphenylphosphine)palladium (0) PPh₃ triphenylphosphine PhSiH₃phenylsilane Py pyridine Py•TFA pyridinium trifluoroacetate TBMEtert-butyl methyl ether TFA trifluoroacetic acid TFFHfluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate THFtetrahydrofuran TM Test Method TsCl tosylchloride

Example 1 Preparation of Compound 22

Preparation of Compound 15 (Step 1)

General Procedure: As shown in Stage 1 above, Compound 15 may beprepared as follows. To an inerted reaction vessel is added Compound 14(1 wt, 1 eq), Pd/C (preferably approximately 0.1-0.2 wt, 0.025-0.050eq), and an appropriate hydrogenation solvent (e.g., a non-aqueous polarsolvent such as THF, preferably a polar protic solvent, more preferablya low molecular weight alcohol; most preferably ethanol or isopropanol)(approximately 6-7 vol). The inert gas in the vessel is replaced withhydrogen, (preferably at a pressure of approximately 1 atmosphere toapproximately 120 psi) and the reaction mixture is stirred at roomtemperature until the reaction is essentially complete (approximately3-8 days). Hydrogen in the reaction vessel is then replaced with aninert gas and the catalyst is removed by filtration (e.g., usingCelite). The filtrate is then concentrated to yield Compound 15(approximately 0.8 wt) as a pale yellow oil. The following proceduresdescribe the hydrogenation of Compound 14 to Compound 15 in ethanol andin isopropanol.

Step 1(a1): Hydrogenation in Ethanol: To an inerted round bottom flaskequipped with a magnetic stirring device was added Compound 14 (3.00 g,1.0 wt, 1.0 eq), 10% Pd/C (Degussa type E101 NE/W, 0.300 g, 0.10 wt,0.024 eq, Aldrich), and ethanol (20.0 mL, 6.7 vol). The inert gas in thevessel, nitrogen, was replaced with hydrogen (1 atmosphere) and thereaction mixture was stirred at room temperature until the reaction wasfound complete by TLC analysis (approximately 4 days). Hydrogen in thereaction vessel was then replaced with nitrogen, the catalyst wasremoved by filtration on Celite and a small amount of ethanol was usedas rinse.

Step 1(a2) Compound 15 may generally be used in situ to prepare Compound22. For characterization purposes, the Compound 15/ethanol filtrate wasconcentrated to yield Compound 15 (2.5 g, 0.83 wt) as a pale yellow oil,redissolved in a minimum amount of 10% methanol/dichloromethane, loadedonto a silica gel column (KP-Sil, 13.0 wt), and eluted with 10%methanol/dichloromethane. A few pure fractions were combined andconcentrated in vacuo to give purified Compound 15 (0.1 wt).

Analytical Data for purified Compound 15: ¹H NMR (400 MHz, CDCl₃) δ:5.03-5.10 (m, 1H), 3.63 (dd, J=10, 5 Hz, 1H), 3.42-3.52 (m, 3H),3.34-3.39 (m, 2H), 3.02-3.07 (m, 1H), 2.29 (t, J=7 Hz, 3H), 1.73-1.94(m, 6H), 1.48-1.66 (m, 4H), 1.20-1.36 (m, 26H), 0.89 (t, J=7 Hz, 6H).ESI-MS (M+H)⁺ Theoretical calculation for C₂₅H₅₂NO₄: 430.4; actualresult: 430.4.

Step 1(b1) Hydrogenation in Isopropanol: To an inerted 12-L glass flaskwere charged Compound 14 (2.0 kg, 1.0 wt) and isopropanol (8.0 kg, 4.0wt), and the mixture was stirred until the Compound 14 was dissolved. Toa 5-gallon inerted reactor equipped with mechanical stiffing werecharged Pd/C (Johnson Matthey type A402028-10, 10% Pd/C, 50% wet; 0.411kg, 0.206 wt, 0.05 eq) and the Compound 14/isopropanol solution.Isopropanol (1.1 kg, 0.55 wt) was used to rinse residual Compound 14into the 5-gallon reactor. The 5-gallon reactor was then pressurizedwith nitrogen to 10-15 psi while stirring at ˜750 rpm. Nitrogen wasvented while stirring at ˜250 rpm. Thenitrogen-pressurization-and-venting cycle was repeated two more times.The reactor was pressurized with hydrogen (grade 5.0) to 25 psi whilestiffing at ˜750 rpm, and the hydrogen was then vented while stiffing at˜250 rpm. The reactor was then pressurized with hydrogen to 120 psiwhile stiffing at ˜750 rpm. The reaction mixture was then stirred atambient temperature for 65 h, after which the hydrogen was vented. Thereactor was then pressurized with nitrogen to 10-15 psi, after which thenitrogen was vented. The nitrogen-pressurization-and-venting process wasrepeated two more times. HPLC monitoring showed a complete reaction. Thecatalyst was then removed by filtration through two CUNO filter housings(a CUNO CTG Klean 1.0 μm filter cartridge and a CUNO CTG Klean 0.2 μmfilter cartridge connected in series) that had been pre-washed withisopropanol (3.9 kg, 1.95 wt). The reactor was rinsed with isopropanol(5.0 kg, 2.5 wt) and the resultant rinse solution was filtered throughthe CUNO housings. The filtrates were combined and produced an Compound15/isopropanol colorless clear solution (15.8 kg, 7.9 wt). Approximately15 kg (7.5 wt) (=0.95×15.8 and 7.9, respectively) of this solution wasconcentrated at reduced pressure to 6.55 kg (3.28 wt), and used in theStep 2a without purification.

Preparation of Compound 22 (Step 2a)

General Procedure: As shown in Stage 2 above, the following generalprocedure may be used to prepare Compound 22 (all weights, equivalentsand volumes are relative to the mass of Compound 14 used in Step 1):Into an inerted glass reactor equipped with a stiffing device is chargedcarbonyldiimidazole (CDI) (0.34 wt, 1.1 eq) and anhydrous acetonitrile(7.4 wt). Stiffing is initiated and the mixture is cooled to about 0° C.3-oxo-tetradecanoic acid (0.514 wt, 1.1 eq) is added and stirring iscontinued while the temperature is maintained at about 0° C. Theimidazolide formation is monitored by HPLC. When determined complete,the Compound 15/IPA solution (3-8 wt) obtained in Step 1 is added,keeping the temperature at around 0° C. The reaction mixture is stirredat around 0° C. and monitored by HPLC. After completion, glacial aceticacid (0.24 wt, 2.1 eq) is added while keeping the reactiontemperature≦15° C. The reaction mixture is brought to about 20° C. andits volume reduced to about 5-7.5 vol by partial solvent evaporation invacuo at 20-25° C., giving a clear orange solution. Heptane (8.5 wt) isadded and the resultant mixture is washed with a solution composed ofNH₄Cl (0.15 wt) and water (5.2 wt). The heptane layer is then washedwith a solution composed of sodium chloride (1.0 wt) and water (3.2 wt).The heptane layer is concentrated in vacuo at about 25-30° C. down toabout 2.2-4.4 vol, then isopropyl acetate (9.0 wt) is added and theresultant solution is concentrated in vacuo at about 25-30° C. to give aclear orange oil (2.2-5.6 vol or 2.0-5.0 wt).

Specific example: The following procedure describes the preparation ofCompound 22 on a scale that involved 95% of the amount of Compound 15produced using 2.0 Kg of Compound 14 in Step 1(b1). Thus for thisexample, 1.9 Kg=1.0 wt. In a 50 L inerted glass reactor equipped with amechanical stirrer was charged carbonyldiimidazole (CDI) (0.65 Kg, 0.34wt, 1.1 eq) and anhydrous acetonitrile (14.1 Kg, 7.4 wt). Stirring wasinitiated and the mixture was cooled to 0° C. Then, 3-oxo-tetradecanoic(0.98 Kg, 0.516 wt, 1.1 eq.; purchased from DSM Pharmaceutical Products,Parsippany, N.J.) was added under continued stiffing, while maintainingthe temperature at about 0° C. The imidazolide formation was monitoredby HPLC. Upon completion, after approximately 10.5 h, the Compound15/IPA solution (6.55 kg, 3.45 wt) obtained in Step 1(b1) was added over1 min and the temperature remained around 0° C. IPA (1.7 Kg, 0.89 wt)was used as rinsing solvent. The reaction mixture was stirred overnightat 0-3° C. HPLC confirmed that the reaction was complete. Glacial aceticacid (0.46 Kg, 0.24 wt, 2.1 eq) was then added while keeping thereaction temperature≦15° C. (The acetic acid addition took placeapproximately 12.25 h after the Compound 15 addition.) The reactionmixture was brought to 20° C. and its volume was reduced to about 13 L(6.8 vol) by partial solvent evaporation in vacuo at 20-22° C., whichproduced a clear orange solution. Heptane (16.2 Kg, 8.53 wt) was addedand the resultant mixture was washed with a solution composed of NH₄Cl(0.29 Kg, 0.15 wt) and water (9.9 Kg, 5.2 wt) followed by washing with asolution composed of sodium chloride (1.9 Kg, 1.0 wt) and water (6.1 Kg,3.2 wt). The heptane layer was concentrated in vacuo at 25-30° C. downto ˜7 L (3.7 vol). Isopropyl acetate (17.0 Kg, 8.95 wt) was then addedand the resultant solution was concentrated in vacuo at 25-30° C., whichproduced a clear orange oil ˜7 L (3.7 vol, Purity (HPLC): 83.8 area %).HPLC conditions for analysis of Compound 22 (HPLC TM1 of Compound 22):

HPLC Column Waters Xterra RP18, 150 × 4.6 mm, 5 μm, Waters catalog186000492 Temperature 35° C. Mobile phase A: 2.0 mL of 28-30% aqueousNH₄OH in 1 L of water, B: 2.0 mL of 28-30% aqueous NH₄OH in 1 L of CH₃CNFlow Rate 1.0 mL/min time, min %-Solvent B Gradient initial  93 15  9318 100 27 100 Injection Volume 10 μL Detection (UV) 225 nm Compound 229.4 min ± 10% Retention time

Compound 22 Crystallization (Step 2b)

General Procedure: Compound 22 may be crystallized from an isopropylacetate/acetonitrile mixture. For example, in a glass vessel, Compound22 (2.2-5.6 vol, based on Compound 14) is dissolved in isopropyl acetate(3.5 wt). The resultant solution is filtered and adjusted to a totalweight of about 7.4 wt with isopropyl acetate. The solution is thentransferred to an appropriately sized jacketed glass reactor equippedwith a stirring device and acetonitrile (5.0 wt) is added. Under inertnitrogen atmosphere, the resultant mixture is cooled to about 5-8° C. toform Compound 22 crystals. The temperature is warmed to about 15° C. todissolve smaller crystals. The temperature is held at 15° C. for about 2h, slowly cooled to about −12° C., and then preferably held at −12° C.for an additional 1 h. Compound 22 is filtered and the solid rinsed witha cold (˜−20° C.) mixture of isopropyl acetate/acetonitrile (1:1 (v/v),1-2 wt). The wet cake is dried to produce Compound 22 as a white solid(approximately 0.9-1.5 wt).

Specific example: The following procedure presents how Compound 22 wascrystallized out on a scale that involved 1.9 Kg of Compound 14 startingmaterial (i.e., 1.9 Kg=1.0 wt): In a glass vessel, the crude Compound 22oil from Step 2a (3.7 vol based on Compound 14) was dissolved inisopropyl acetate (6.7 Kg, 3.5 wt). The resultant solution was filteredand excess solvent was removed in vacuo at 25-30° C. The remainingsolution (7.23 Kg, (3.8 wt)) was then transferred to a 50 L jacketedglass reactor equipped with a mechanical stirrer and the mass adjustedto about 14.0 Kg (7.4 wt) by the addition of isopropyl acetate (6.27 Kg,3.3 wt). Acetonitrile (9.6 Kg, 5.1 wt) was then added. Under inertnitrogen atmosphere, stiffing was initiated and the resultant mixturewas cooled to 7.3° C. to form Compound 22 crystals. The temperature wasthen raised to 15.1° C. to dissolve smaller crystals. The temperaturewas held at 15° C. for about 2 h, slowly cooled to −11.7° C. overnight,and then held at around −12° C. for about 1 h. The resulting compositionwas filtered and the solid was rinsed with a cold (˜−20° C.) mixture ofisopropyl acetate/acetonitrile (1:1 (v/v), 2.6 Kg, 1.4 wt). The wet cakewas dried in vacuo, which produced Compound 22 as a white solid, (1.83Kg, 0.96 wt; Purity: 92.5 area % (HPLC TM1 of Compound 22); Yield: 69%from Compound 14).

Compound 22 Recrystallization (Step 2c)

General Procedure: In an appropriately sized and inerted jacketed glassreactor equipped with a stiffing device are combined Compound 22 (1.0 wt(based on Compound 22)), isopropyl acetate (4.4-4.9 wt) and acetonitrile(3.9-4.5 wt). The resultant mixture is stirred and warmed to 20-25° C.to give a clear solution. The clear solution is cooled to 5-10° C. toform crystals. The temperature is then warmed to 16-20° C. to dissolvesmaller crystals and held at about 17° C. for about 2 h. The temperatureis slowly cooled linearly to about −3 to −8° C. over about 10-11 h.Preferably the temperature is held at −3 to −8° C. for an additional 2h. The solid is filtered and rinsed with a cold (−20° C.) solution ofisopropyl acetate/acetonitrile (1:1 (v/v), 1-3 wt) and dried in vacuo toproduce Compound 22 as a white powder, (approximately 0.8-0.95 wt).

Specific Example: The following procedure describes therecrystallization of Compound 22: 1.79 Kg of Compound 22 from thecrystallization in Step 2b (@92.5% purity=1.65 Kg compound; 1.65 Kg=1.0wt) were combined with isopropyl acetate (8.0 Kg, 4.8 wt) andacetonitrile (7.37 Kg, 4.5 wt) in an inerted 30 L jacketed glass reactorequipped with a mechanical stirrer. The resultant mixture was stirredand warmed to 24.3° C. to give a clear solution. The clear solution wascooled to 10.4° C. to form crystals. Then, to dissolve smaller crystals,the temperature was warmed to 16.2° C. and was held at 16.2-17.3° C. forabout 2 h. The temperature was slowly cooled to −3.4° C. overnight. Thefollowing day, the temperature was held at that temperature for anadditional 20 min. The solid was filtered and rinsed with a cold (—20°C.) solution of isopropyl acetate/acetonitrile (1:1 (v/v), 4.6 Kg, 2.8wt) and dried in vacuo (18-25° C., 5 h), which produced Compound 22 as awhite solid, (1.54 Kg, 0.93 wt; Purity: 99.4% (HPLC TM1 of Compound 22);Yield: 92.8%).

Analytical Data for Compound 22: ¹H NMR (400 MHz, CDCl₃) δ: 7.24 (d, J=7Hz, 1H), 5.05-5.14 (m, 1H), 4.03-4.11 (m, 1H), 3.72 (dd, J=5, 5 Hz, 2H),3.42-3.60 (m, 3H), 3.41 (s, 2H), 3.26-3.38 (m, 1H), 2.53 (t, J=7 Hz,3H), 2.29 (t, J=7 Hz, 3H), 1.79-1.89 (m, 1H), 1.48-1.77 (m, 7H),1.19-1.38 (m, 42H), 0.88 (t, J=7 Hz, 3H). ESI-MS (M+H)⁺ Theoreticalcalculation for C₃₉H₇₆NO₆: 654.6; actual result: 654.6.

Example 2 Solid State Characterization of Crystalline Compound 22

A. Powder X-Ray Diffraction

Using a glass plate, on the Scintag Diffractometer, data were collectedunder normal powder diffraction conditions, with 2-theta range of 3-40degrees, using copper radiation. No background correction was applied.FIG. 2 shows the PXRD pattern of crystalline Compound 22. The PXRDpattern shows peaks at 10.3±0.2°2Θ, 12.3±0.2°2Θ, 14.5±0.2°2Θ, 15.3±0.2°2Θ, 16.2±0.2°2Θ, 17.8±0.2°2Θ, 22.2±0.2°2Θ, 22.9±0.2°2Θ, 23.8±0.2°2Θ,25.3±0.2° 2Θ, and 26.8±0.2°2Θ. Crystalline Compound 22 may becharacterized by a subset of the peaks shown in FIG. 2. For example, thefollowing peaks are characteristic of crystalline Compound 22:12.3±0.2°2Θ, 14.5±0.2°2Θ, 16.2±0.2°2Θ, 17.8±0.2°2Θ, 22.2±0.2°2Θ, and23.8±0.2°2Θ. Other combinations of the peaks listed or shown in FIG. 2may also be used to identify Compound 22.

TABLE 1 Measurement conditions X-ray diffractometer: Scintag Target: CuDetector: Scintillation Counter Tube voltage: 40 kV Tube current: 20 mASlit: DS 1°, RS 0.3 mm, SS 1° Scan speed: 2°/min Sampling width: 0.02°Scan range: 3 to 40° Sample holder: glass holder Goniometer: horizontalgoniometer Filter: not used

B. DSC Characterization

Solid-state characterization of crystalline Compound 22 was determinedby Differential Scanning calorimetry (DSC, aluminum pan technique). TheDSC was run with a 2920 DSC V2.5F calorimeter heating to 150° C. at 10°C./min with an aluminum pan under a nitrogen purge of 50 mL/min using a2.91 mg sample of crystalline Compound 22. FIG. 3 shows the thermogramof crystalline Compound 22 with melting at 41° C. (onset temp.).

A melting point experiment was also conducted using an ElectrothermalMel.Temp Apparatus, with a Fluke 51 II Digital Thermometer. Compound 22(2-3 mg) was loaded in a capillary tube (1.5-1.8×90 mm, Kimble ProductKIMAX-51, part no. 34505). The observed melting point was 41-42° C.

Example 3 Compound 25 Synthesis and Seed Crystals Formation

A. Compound 25 Synthesis and Crude Crystals Preparation (Step 3a)

Synthesis of Compound 25: Into an inerted jacketed glass reactorequipped with a mechanical stirrer were added Compound 24(N,N′-bis(2-hydroxyethyl)urea; 8.00 g, 1.00 wt, 1.00 eq; purchased fromvendor Mitsui & Co. (USA), Inc., New York, N.Y.; manufacturer YoyuLabs), pyridinium trifluoroacetate (0.5 g, 0.063 wt, 0.05 eq) andacetonitrile (59.7 g, 7.5 wt). Stirring was initiated and allyltetraisopropylphosphorodiamidate (35.0 g, 4.4 wt, 2.24 eq; purchasedfrom Digital Specialty Chemicals, Inc., Dublin, N.H.) was added. Theresultant mixture was then stirred at 20-25° C. overnight (17 h). HPLCmonitoring revealed a complete reaction. The temperature was cooled to0° C., and a precipitate appeared. The temperature was slowly warmed to16° C. over 2 h to dissolve most of the solid that had formed. Themixture was then stirred at 16° C. for 1.5 h, cooled to −17.4° C. at arate of 5° C./h, and stirred overnight at that temperature, producing athick suspension. The temperature was then raised to 16° C. over 2.3 hand maintained at 16° C. for 2.25 h. The temperature was dropped to 3.5°C. over 1.2 h and then quickly (approx. 5-10 min) warmed to 10° C. Thetemperature was held at 10° C. for 2 h and was cooled overnight at arate of about 1.5° C./h to a final temperature of −16° C. The solid thathad formed was filtered, washed with cold acetonitrile (2×6.3 g, 2×0.79wt), and dried under vacuum. Compound 25 (19.7 g, 2.46 wt) was obtainedas a white solid.

B. Compound 25 Recrystallization (Step 3b)

Into an inerted jacketed glass reactor equipped with a mechanicalstirrer was added Compound 25 from Step 3a (19.5 g, 1.00 wt) andacetonitrile (55.0 g, 2.8 wt). The resultant mixture was stirred at20-25° C. for 15 min. The temperature was cooled to 10° C., at whichpoint stirring became difficult. The temperature was raised to 20° C.and maintained there for 2 h. Cooling to 10° C. was resumed at a rate ofabout 2° C./h. Cooling was continued to 1° C. a rate of about 3° C./h,followed by cooling to −19° C. at a rate of about 5° C./h. The solidthat formed was filtered, washed with cold acetonitrile (32 g, 1.6 wt),and dried under vacuum. Compound 25 (16.06 g, 0.82 wt) was obtained as awhite solid.

C. Compound 25 Seed Crystals Formation (Step 3c)

The following procedure can be and was used to prepare the seed crystalsreferenced in Step 4a and Step 4b, below: Into an inerted jacketed glassreactor equipped with a mechanical stirrer was added Compound 25 fromStep 3b (5.59 g, 1.00 wt) and a solution composed of heptane and TBME(9:1 (v/v), 120 mL, 21.5 vol). The resultant mixture was stirred at20-25° C., and more TBME (3.0 mL, 0.54 vol) was added to completelydissolve Compound 25. The temperature was reduced to 10° C. over about1.5 h, forming a white precipitate. The temperature was warmed to 16° C.and maintained there for about 2 h. The reactor was cooled at a rate ofabout 2° C./h to 0° C., followed by cooling at a rate of about 3° C./hto −18.8° C., then maintained at about −18.8. This cooling profile wascarried out overnight. The solid that formed was filtered, washed with acold solution composed of heptane and TBME (9:1 (v/v), 16 g, 2.9 wt),and dried under vacuum. Compound 25 (5.23 g, 0.936 wt; Purity: 94.7%(area percent)) was obtained as a white solid. HPLC conditions foranalysis of Compound 25 (HPLC TM2 of Compound 25)

HPLC Column Phenomenex Luna phenyl-hexyl, 250 × 4.6 mm, 5 μm. Catalog #00G-4257-E0. Mobile phase A = water B = CH₃CN Flow Rate 1.0 mL/min time,min %-Solvent B Gradient initial  2 40.0  26 75.0 100 85.0 100 InjectionVolume 3 μL Detection (UV) 200 nm Compound 25 30 min ± 10% Retentiontime

Example 4 Preparation of Compound 25

Preparation of Compound 25 (Step 4a)

General Procedure to prepare Compound 25: (Note: 1.0 wt herein refers tothe mass of Compound 24 used as starting material to produce a batch ofCompound 25). In an appropriately sized and inerted jacketed glassreactor equipped with a stiffing device are combined urea Compound 24(1.0 wt, 1.0 eq), pyridinium trifluoroacetate (0.07 wt, 0.05 eq) andanhydrous acetonitrile (7.0 wt). Stirring is initiated and allyltetraisopropylphosphorodiamidite (4.4 wt, 2.3 eq) is added. The reactionis stirred at 18-26° C. for several hours until complete. The resultantmixture is cooled to approximately 9-11° C., preferably seeded withcrystalline Compound 25 (0.005 wt; the preparation of the Compound 25seed crystals is discussed in Example 3, above) and the temperaturemaintained for about 2 h. The mixture is then cooled to about 0 to −15°C. at a rate of about 1.5° C./hour, followed by cooling to about −20° C.at a rate of about 2.5° C./hour. After holding the temperature stablefor about 2 hours, the solid is filtered under a nitrogen atmosphere,washed with cold (−20° C.) anhydrous acetonitrile (2-3 wt) and driedunder nitrogen. Compound 25 (about 2.3-2.5 wt) is thus produced as awhite solid. Compound 25 is preferably stored under dry nitrogen at lowtemperature (e.g., −20° C.) until purification.

Specific example: The procedure below describes the synthesis ofCompound 25 starting from Compound 24 (0.50 Kg, referred herein as 1.0wt): In a 30 L inerted jacketed glass reactor equipped with a mechanicalstirrer were combined urea Compound 24 (0.50 Kg, 1.0 wt, 1.0 eq),pyridinium trifluoroacetate (0.035 Kg, 0.07 wt, 0.05 eq) and anhydrousacetonitrile (3.5 Kg, 7.0 wt). Stirring was initiated and allyltetraisopropylphosphorodiamidite (2.22 Kg, 4.4 wt, 2.3 eq) was added.The reaction was stirred at 18.4-25.6° C. It was monitored by HPLC,which confirmed that the reaction was complete after 18 h. The resultantmixture was cooled to 11.1° C. and the temperature was kept at 9.3-11.1C for about 50 min. The mixture was seeded with crystalline Compound 25(1.9 g, 0.004 wt; the preparation of the Compound 25 seed crystals isdiscussed in Example 3, above) and kept in the 9-11° C. temperaturerange for about 2 h. The mixture was then cooled to −7.9° C. at a rateof about 1.5° C./hour. The cooling rate was then accelerated to about2.5° C./hour and the temperature was brought to −18° C. The temperaturewas held at −18 to −19.2° C. for about 2 h, after which the solid thathad formed was filtered under a nitrogen atmosphere, washed with cold(˜−20° C.) anhydrous acetonitrile (1.5 Kg, 3.0 wt), and dried under astream of nitrogen. Compound 25 (1.18 Kg, 2.36 wt; Purity: 93.6% areapercent (HPLC TM2 of Compound 25)) was obtained as a white solid and wasstored under nitrogen at about −20° C. until purification.

Compound 25 Recrystallization (Step 4b)

General Procedure for the recrystallization of Compound 25: In anappropriately sized and inerted glass reactor equipped with a stiffingdevice, urea di-phosphoramidite Compound 25 (1.0 wt, 1.0 wt hereinrefers to the mass of crude Compound 25 precipitated from the reactionthat produced it) is dissolved in TBME (1.5-2.0 wt). Insolubleparticulates are removed by filtration. The filtrate is transferred toan appropriately sized and inerted jacketed glass reactor equipped witha stiffing device. Heptane (13.2 wt) is added, and the resultantsolution is cooled to about 9-11° C. and stirred in that temperaturerange for about 1.5-2.5 h. A white suspension is formed (seeding may beuseful to facilitate Compound 25 crystallization; the preparation of theCompound 25 seed crystals is discussed in Example 3, above). The mixtureis cooled to between about 0 to about −15° C. at a rate of about 1.5°C./hour, followed by cooling to about −20° C. at a rate of about 2.5°C./hour. After holding the temperature stable for about 2 hours thesolid is filtered, washed with a cold solution (˜−20° C.) composed ofheptane and TBME (7:1 (vol/vol), 1.3-1.5 wt) and dried under a stream ofnitrogen. Compound 25 is obtained as a white solid (approximately0.5-0.6 wt). The filtrate is concentrated in vacuo at about 20-30° C.and recrystallized according to the procedure just described. Thus, asecond crop of Compound 25 (0.2-0.3 wt) may also be obtained.

Specific example: The following procedure describes therecrystallization of Compound 25 (1.18 Kg, referred herein as 1.0 wt).In a 22 L Rotavap glass bulb under nitrogen atmosphere, ureadi-phosphoramidite Compound 25 from Step 4a (1.18 Kg, 1.0 wt) wasdissolved in TBME (1.81 Kg, 1.5 wt). Insoluble particulates were removedby filtration, using TBME (0.60 Kg, 0.50 wt) as a rinse. The filtratewas transferred to an inerted 30 L jacketed glass reactor equipped witha mechanical stirrer. Heptane (15.54 Kg, 13.2 wt) was added, theresultant solution was cooled to 10.3° C. Compound 25 started toprecipitate about 5 min later. Stirring was continued at 9-11° C. forabout 130 min. The mixture was then cooled at a rate of about 1.5°C./hour overnight to a temperature of about −15° C. The next day coolingwas continued at a rate of about 1.5° C./hour to a temperature of −17.2°C., after which the temperature was held for about 2 hours between −17.0to −20° C. The solid that was formed was then filtered, washed with acold solution (˜−20° C.) composed of heptane and TBME (7:1 (vol/vol),1.56 Kg, 1.3 wt), and dried under a stream of nitrogen for about 22.5 h.Compound 25 was obtained as a white solid (0.675 Kg, 0.57 wt, Purity:93.9% area percent (HPLC TM2 of Compound 25); Yield: 36.0% from Compound24). The filtrate was concentrated in vacuo at 20-30° C. andrecrystallized according to the procedure just described. Thus, a secondcrop of Compound 25 was obtained as a white solid (0.225 Kg, 0.19 wt,Purity: 92.6% area percent (HPLC TM2 of Compound 25); Yield: 11.8% fromCompound 24).

Analytical Data for Compound 25: ¹H-NMR (400 MHz, CDCl₃) δ: 5.90-6.01(m, 2H), 5.30 (d, J=17 Hz, 2H), 5.16 (d, J=10 Hz, 2H), 4.79 (t, J=5 Hz,2H), 4.06-4.26 (m, 4H), 3.66-3.79 (m, 4H), 3.54-3.66 (m, 4H), 3.33-3.47(m, 4H), 1.18 (d, J=7 Hz, 24H). ESI-MS (M+Na)⁺ Theoretical calculationfor C₂₃H₄₈N₄NaO₅P₂: 545.3; actual result: 545.4

Example 5 Solid State Characterization of Crystalline Compound 25

A. Powder X-ray Diffraction

The powder X-ray diffraction (PXRD) pattern was obtained using the sameprocedure as described above in Example 2A. FIG. 4 shows the PXRDpattern of recrystallized Compound 25. The PXRD pattern shows peaks at6.6±0.3°2Θ, 13.2±0.3°2Θ, 14.0±0.3°2Θ, 17.3±0.3°2Θ, 19.4±0.3°2Θ,21.8±0.3°2Θ, 22.4±0.3°2Θ, 23.6±0.3°2Θ and 27.2±0.3 °2Θ. CrystallineCompound 25 may be characterized by a subset of the peaks shown in FIG.4. For example, the following peaks are characteristic of crystallineCompound 25: 6.6±0.3°2Θ, 14.0±0.3°2Θ, 17.3±0.3°2Θ, 19.4±0.3°2Θ. Othercombinations of the peaks listed or shown in FIG. 2 may be also used toidentify Compound 25.

B. DSC Characterization

Solid-state characterization of crystalline Compound 25 was determinedby Differential Scanning calorimetry (DSC, capillary technique). The DSCwas run with a 2920 DSC V2.5F calorimeter heating to 100° C. at about10° C./min under nitrogen with an aluminum pan using a 1.88 mg sample ofcrystalline Compound 25. FIG. 5 shows the thermograms of crystallineCompound 25, where the sample of Compound 25 melted at 65° C. (onsettemp.).

A melting point experiment was also conducted using an ElectrothermalMelt Temp Apparatus, with a Fluke 51 II Digital Thermometer. Compound 25(2-3 mg) was loaded in a capillary tube (1.5-1.8×90 mm, Kimble ProductKIMAX-51, part no. 34505). The observed melting point was 65-67° C.

C. Single Crystal X-ray Diffraction

Compound 25, 150 mg, was dissolved in acetonitrile, 1 mL, at about 25°C. The solution was cooled to about 0° C. over about 425 minutes andthen held for about 1000 minutes. A single crystal suitable for x-raydiffraction was obtained. The crystal structure exhibited a colorlessneedle crystal structure having dimensions of 0.18×0.06×0.06 mm. Thecrystal was mounted on a 0.2 nm nylon loop using a small amount ofparatone oil.

Data were collected using a Bruker SMART CCD (charge coupled device)based diffractometer equipped with an Oxford Cryostream low-temperatureapparatus operating at about 193° K. Data were measured using omegascans of 0.3° per frame for about 45 seconds, such that a hemisphere wascollected. A total of 1271 frames were collected with a maximumresolution of 0.76 Å. The first 50 frames were recollected at the end ofdata collection to monitor for decay. Cell parameters were retrievedusing SMART software (SMART V 5.625 (NT) Software for the CCD DetectorSystem; Bruker Analytical X-ray Systems, Madison, Wis. (2001)), andrefined using SAINT on all observed reflections. Data reduction wasperformed using the SAINT software (SAINT V 6.22 (NT) Software for theCCD Detector System Bruker Analytical X-ray Systems, Madison, Wis.(2001)), which corrects for Lp and decay. Absorption corrections wereapplied using the SADABS multiscan technique (see SADABS, Program forabsorption corrections using Siemens CCD, Blessing, R. H. Acta Cryst.A51 1995, 33-38). The structures were solved by the direct method usingthe SHELXS-97 program (see Sheldrick, G. M. SHELXS-90, Program for theSolution of Crystal Structure, University of Göttingen, Germany, 1990),and refined by the least squares method on F², SHELXL-97, (seeSheldrick, G. M. SHELXL-97, Program for the Refinement of CrystalStructure, University of Göttingen, Germany, 1997), incorporated inSHELXTL-PC V 6.10, (SHELXTL 6.1 (PC-Version), Program library forStructure Solution and Molecular Graphics; Bruker Analytical X-raySystems, Madison, Wis., 2000). The structure showed signs of beingtwinned, evidenced by the initial cell parameters and peak profiles. Theuse of the program Cell_Now showed that there is a two fold rotation,minor twin component that is orientated along and rotated about thereciprocal axis—0.010 1.000-0.040 and real axis 0.001 1.000-0.002. Thetwin data was integrated and refined. The percentage of twin refinementwas found to be less than 1.5%.

The structure was solved in the space group P2₁/c (#14). Allnon-hydrogen atoms were refined anisotropically. Hydrogens werecalculated by geometrical methods and refined as a riding model. Thecrystal used for the diffraction study showed no decomposition duringdata collection. All drawings are done at 50% ellipsoids. An ORTEPdrawing of crystalline Compound 25 with various atom labeling is shownin FIG. 6. FIG. 7 shows the crystal packing diagram along the c-axis.The crystal data and structure refinement parameters are reported inTable 2.

TABLE 2 Crystal data and structure refinement for crystalline Compound25. Empirical formula C₂₃H₄₈N₄O₅P₂ Formula weight 522.59 Temperature193(2) °K Wavelength 0.71073 Å Crystal system Monoclinic Space group P2(1)/c Unit cell dimensions a = 27.134(14) Å α = 90°. b = 12.135(6) Å β =93.156(10)°. c = 9.332(5) Å γ = 90°. Volume 3068(3) Å³ Z 4 Density(calculated) 1.131 mg/m³ Absorption coefficient 0.177 mm⁻¹ F (000) 1136Crystal size 0.18 × 0.06 × 0.06 mm³ Theta range for data collection 1.50to 22.60° Index ranges −22 ≦ h ≦ 29, −13 ≦ k ≦ 13, −8 ≦ l ≦ 10Reflections collected 11908 Independent reflections 4010 [R (int) =0.1578] Completeness to theta = 22.60° 99.0% Absorption correctionMultiscan Max. and min. transmission 0.9895 and 0.9689 Refinement methodFull-matrix least-squares on F² Data/restraints/parameters 4010/179/339Goodness-of-fit on F² 0.970 Final R indices [I > 2σ (I)] R1 = 0.1031,wR2 = 0.2584 R indices (all data) R1 = 0.1851, wR2 = 0.3034 Largestdiff. peak and hole 0.347 and −0.233 e · Å⁻³

A simulated XRPD pattern based on the single crystal data for Compound25 is shown in FIG. 8. The powder diffraction data was simulated fromthe single crystal intensity data observed using the program XPOW.(XPOW, Simulated Powder Diffraction Pattern, Version 5.101, Bruker-AXS,1997-1998.) Key parameters for the calculation include the wavelength,used copper wavelength of 1.54 {acute over (Å)}, and the cell parametersretrieved from the final refinements. The line width and intensity isdependant on the equation:

Height=intensity/[1+4*x*x(w=v*tan(θ))]

x=2θ/2θ₀ where 2θ₀ is the Bragg angle for the reflection. The variablesw and v are lineshape parameters. For these calculations 0.02 was usedfor both w and v. The table below lists the nine peaks with the highestintensity.

Peak Position, °2Θ A 13.9 B 13.4 C 17.4 D 19.6 E 9.8 F 22.0 G 13.1 H 6.5I 23.5

Example 6 Preparation of Compound 26

Preparation of Compound 26 (Step 5a)

General Procedure for the synthesis of Compound 26: (Note: 1.0 wt hereinrefers to the mass of Compound 22 used as starting material to produce abatch of Compound 26). In an appropriately sized and inerted glassreactor equipped with a stirring device are charged Compound 22, forexample Compound 22 obtained from Steps 2b-2c, preferably Step 2c, (1.0wt, 1.0 eq) and Compound 25, for example Compound 25 obtained from anyof Steps 4a-4-b, preferably Step 4b, (0.42 wt, 0.52 eq). Anhydrousacetonitrile (5.0 wt) and anhydrous heptane (1.4 wt) are added andstiffing is initiated. The mixture is warmed to about 20-25° C. in orderto dissolve all solids. Acetic acid (0.12 wt, 1.3 eq) is slowly added,maintaining the temperature at about 20-25° C. The reaction is stirredat about 20-25° C. for several hours until complete. Anhydrous heptane(8.0 wt) is charged in the reactor and the temperature is cooled toabout 0-5° C. H₂O₂ 30% (wt/wt) in water (0.17 wt, 1.1 eq) is slowlyadded over about 0.5 h, maintaining the temperature at ≦5° C. Thereaction is stirred at ≦5° C. until complete. The reaction is thencooled to about 0° C. and residual peroxides are quenched by theaddition of an aqueous solution of sodium thiosulfate pentahydrate (1/1(w/w), 0.26 wt, 0.34 eq). Stirring is continued at about 0-4° C. untilthe heptane layer returns a negative test for peroxides. The temperatureis raised to room temperature and then the salts are filtered off andrinsed with heptane (2.3 wt). The resultant filtrate is charged into anew glass reactor and layers are allowed to separate for about 15 min.Three layers are formed, with the bottom two layers (aqueous andacetonitrile) being drained and discarded. The top, heptane layer iswashed twice with acetonitrile (1.8 wt) and concentrated to dryness atabout 18-25° C. Crude Compound 26 (1.2-1.5 wt) is obtained as a thick,slightly amber oil.

Specific example: The following procedure describes the preparation ofCompound 26 on a scale that involved 0.565 Kg of Compound 22 with apurity of 99.4%. (The term “1.0 wt” in this example refers to 0.56 Kg,which was the actual mass of Compound 22 used as starting material toproduce Compound 26.) In a 30 L inerted jacketed glass reactor equippedwith a mechanical stirrer were charged Compound 22 from Step 2c (0.56 Kg(=0.565×0.994), 1.0 wt, 1.0 eq) and Compound 25 from Step 4b (0.248 Kg,Purity: 93.9%, 0.42 wt, 0.52 eq). Anhydrous acetonitrile (2.8 Kg, 5.0wt) and anhydrous heptane (0.79 Kg, 1.4 wt) were added and stirring wasinitiated. The mixture was stirred and warmed to about 20-22° C. inorder to dissolve all solids. Acetic acid (0.068 Kg, 0.12 wt, 1.3 eq)was added over about 5 min while the temperature was maintained at about22-24° C. The reaction was stirred at about 20-24° C. for about 24 h,and completion was verified by HPLC. Anhydrous heptane (4.49 Kg, 8.0wt)) was charged into the reactor and the temperature was cooled toabout 0-5° C. H₂O₂ 30% (wt/wt) in water (0.106 Kg, 0.19 wt, 1.1 eq) wasslowly added over about 26 min and the temperature remained between 0-2°C. The reaction was stirred for about 3.5 h while the temperature wasmaintained between −1 to 2° C. The oxidation completion was confirmed byHPLC. Residual peroxides were quenched at about 0° C. by the addition ofan aqueous solution of sodium thiosulfate pentahydrate (1/1 (wt/wt),0.146 kg, 0.26 wt, 0.34 eq). Stirring was continued overnight (17.25 h)at about 0-2° C. The heptane layer was tested for the presence ofperoxides (EM Quant peroxides test, EM Science, Gibbstown, N.J.),returning a negative result. The temperature was raised to about 20° C.Salts were filtered off and rinsed with heptane (1.26 Kg, 2.25 wt). Theresultant filtrate was charged into a new glass reactor and layers wereallowed to separate for about 15 min. Three layers were formed, and thebottom two layers (aqueous and acetonitrile) were drained and discarded.The top, heptane layer was washed twice with acetonitrile (1.0 Kg, 1.8wt) and concentrated to dryness in vacuo at about 18-23° C. CrudeCompound 26 (0.79 Kg, <1.4 wt; Purity 91.1% area percent) was obtainedas a thick oil, slightly amber, containing 5.6% of residual heptane.HPLC conditions for analysis of Compound 26 (HPLC TM3 of Compound 26):

HPLC column Waters Symmetry 300 ™ C18, 250 × 4.6 mm 5 μm Temperature 55°C. Flow rate 1.0 mL/min Mobile Phase A 20 mL 85% phosphoric acid in 1000mL water Mobile Phase B 20 mL 85% phosphoric acid in 1000 mL ethanoltime, min %-Solvent B Gradient initial  30 15  92 35  92 45 100 60 100Injection Volume 20 μL Detection UV = 215 nm Compound 26 33.1 min ± 10%Retention Time

Compound 26 Purification (Step 5b)

General Procedure for the purification of Compound 26: (Note: 1.0 wtherein refers to the amount of Compound 22 used to produce crudeCompound 26). Crude Compound 26, e.g., as obtained from the Step 5a(approximately 1.2-1.5 wt) is dissolved in a mixture of isopropanol andethyl acetate (1/99 (vol/vol), 2 wt) and the resultant solution isloaded onto a Biotage silica gel column (approximately 10 wt of SiO₂)pre-equilibrated with a mixture of isopropanol and ethyl acetate (1/99(vol/vol), approximately 55 wt). Separation is performed usingisopropanol/ethyl acetate (3/97 (vol/vol), approximately 80 wt) followedby isopropanol/ethyl acetate (10/90 (vol/vol), approximately 110 wt).Preferably, around thirty-five fractions of ˜4 wt are collected,including, e.g., fractions containing ≧1% of the theoretical yield ofCompound 26 (≧0.0127 wt of Compound 26, (HPLC assessment)). Desiredfractions are combined and concentrated in vacuo at about 20-30° C. toyield a clear oil. The clear oil is dissolved in heptane (approximately7.0 wt) and the resultant solution is concentrated to dryness in vacuoat about 20-30° C. The heptane treatment is repeated once again andCompound 26 (1.0-1.15 wt; Yield: 75-85%) is obtained as a colorlessclear oil.

Specific Example: Purification of crude Compound 26 (0.79 Kg). Note: 1.0wt herein refers to the amount of Compound 22 (0.56 Kg) used to producecrude Compound 26. Crude Compound 26 from Step 5a (0.79 Kg, <1.4 wt) wasdissolved in a mixture of isopropanol and ethyl acetate (1/99 (vol/vol),1.18 Kg, 2.1 wt) and the resultant solution was loaded onto a BiotageKP-Sil 150L (5.0 Kg of silica, 8.9 wt.) column pre-equilibrated with amixture of isopropanol and ethyl acetate (1/99 (vol/vol), 31.5 Kg, 56.3wt). The column was run with isopropanol/ethyl acetate (3/97 (vol/vol),43.9 Kg, 78.4 wt) followed by isopropanol/ethyl acetate (10/90(vol/vol), 62.3 Kg, 111.3 wt). Thirty-six fractions of approximately 2.3kg (approximately 4.1 wt) each were collected. Fractions containing ≧1%of the theoretical yield of Compound 26 (≧7.1 g of Compound 26), in thiscase fractions 7 to 31, were combined and concentrated in vacuo at21-28° C. to a clear oil. The clear oil was dissolved in heptane (3.9Kg, 7.0 wt) and the resultant solution was concentrated to dryness invacuo at 24-30° C. The heptane treatment was repeated once again andCompound 26 (0.60 kg, 1.07 wt; Purity: 93.4% (HPLC TM3 of Compound 26);Yield=78.8%) was obtained as a colorless clear oil.

Analytical Data for Compound 26: ¹H-NMR (400 MHz, CDCl₃) δ: 7.4-7.57 (m,2H), 6.06 (t, J=5 Hz, 2H), 5.84-6.04 (m, 2H), 5.36 (d, J=17 Hz, 2H),5.25 (d, J=10 Hz, 2H), 5.02 (m, 2H), 4.54 (m, 4H), 4.24 (m, 2H),3.96-4.20 (m, 8H), 3.38-3.55 (m, 16H), 2.52 (t, J=7 Hz, 4H), 2.28 (t,J=7 Hz, 4H), 1.68-1.91 (m, 4H), 1.47-1.67 (m, 12H), 1.18-1.37 (m, 84H),0.89 (t, J=7 Hz, 18H). ESI-MS (M+Na)⁺ Theoretical calculation forC₈₉H₁₆₈ N₄NaO₁₉P₂: 1682.2; actual result: 1682.3.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the spirit and scope of the present invention. Allsuch modifications are intended to be within the scope of the claimsappended hereto.

All patents and publications cited above are hereby incorporated byreference.

The claimed invention is:
 1. A compound of formula (3):

wherein: R¹ is a C₅-C₁₅ alkyl group, a C₅-C₁₅ alkenyl group, or a C₅-C₁₅alkynyl group; R² is a C₅-C₁₅ alkyl group, a C₅-C₁₅ alkenyl group, or aC₅-C₁₅ alkynyl group; and R³ is a C₅-C₁₅ alkyl group, a C₅-C₁₅ alkenylgroup, or a C₅-C₁₅ alkynyl group.
 2. A compound according to claim 1,wherein R¹ is a C₅-C₁₂ alkyl group, R² is a C₇-C₁₄ alkyl group, and R³is a C₇-C₁₄ alkyl group.
 3. A compound according to claim 2, wherein R¹is a C₇ alkyl group, R² is a C₁₁ alkyl group, and R³ is a C₁₁ alkylgroup.
 4. A compound according to claim 3, wherein R¹ is an n-heptylgroup, R² is an n-undecyl group, and R³ is an n-undecyl group.
 5. Acompound according to claim 1, where at least one of R¹, R² and R³ isdifferent from the others.
 6. A compound according to claim 1, havingthe stereochemistry of formula (3a):


7. A compound according to claim 6, wherein the compound is compound 22:


8. A process for making a compound of formula (3) by a reaction of acompound of formula (1) with a compound of formula (2) as shown below

wherein: R¹ is a C₅-C₁₅ alkyl group, a C₅-C₁₅ alkenyl group, or a C₅-C₁₅alkynyl group; R² is a C₅-C₁₅ alkyl group, a C₅-C₁₅ alkenyl group, or aC₅-C₁₅ alkynyl group; R³ is a C₅-C₁₅ alkyl group, a C₅-C₁₅ alkenylgroup, or a C₅-C₁₅ alkynyl group; and X is a leaving group.
 9. A processaccording to claim 8, wherein R¹ is a C₅-C₁₂ alkyl group, R² is a C₇-C₁₄alkyl group, R³ is a C₇-C₁₄ alkyl group, and X is OH, Cl, F,imidazolidyl, trimethyl acetoxy, ethyl carbonate, methyl carbonate,isobutyl carbonate, or a group of the formula Z:

wherein R⁷ and R³ in formula (2) are identical such that the compound offormula (2) is a symmetrical beta ketoester anhydride.
 10. A processaccording to claim 9, wherein R¹ is a C₇ alkyl group; R² is a C₁₁ alkylgroup; and R³ is a C₁₁ alkyl group.
 11. A process according to claim 10,wherein R¹ is an n-heptyl group; R² is an n-undecyl group; and R³ is ann-undecyl group.
 12. A process according to claim 8, where at least oneof R¹, R² and R³ is different from the others.
 13. A process accordingto claim 8, further comprising the step of hydrogenating a compound offormula Y:

in isopropanol to form a compound of formula (1).
 14. Crystallinecompound 22


15. Crystalline compound 22 according to claim 14, further characterizedby an x-ray powder diffraction pattern having peaks at 12.3±0.2°2Θ,14.5±0.2°2Θ, 16.2±0.2°2Θ, 17.8±0.2°2Θ, 22.2±0.2°2Θ, and 23.8±0.2°2Θ. 16.Crystalline compound 22 according to claim 15, further characterized byhaving a melting point of about 41° C.